Identification of the components, targets, and magnitude of an effective immune response to HIV are important steps toward the development of effective vaccines or immunotherapies. Although patients with normal CD4+ T cell counts and low levels of plasma virus are a heterogeneous group, a small subgroup of patients with truly non-progressive HIV infection and restriction of virus replication likely holds important clues to the basis of an effective immune response to HIV. A small subpopulation (fewer than 0.8% of HIV infected individuals) shows no signs of progression over a 10-year period. We have assembled a stringently defined cohort of such patients, termed long-term nonprogressors (LTNP) or elite controllers. Many of these patients have been infected for 20 years with no CD4+ T cell decline without taking antiretroviral therapy, and maintain plasma viral RNA below 50 copies per milliliter. Cells from these patients are being used to systematically dissect the mechanisms of immune mediated restriction of virus replication. HIV-specific T cell responses of these patients have been studied in extreme detail. Prior work has indicated that there is a dramatic association with the HLA B*5701 allele and that the immune response is highly focused on peptides restricted by this allele. More recently we have found that this focus is specific to HIV, and is not found in the response to other pathogens such as Hepatitis C virus or Cytomegalovirus. LTNP patients do not differ in the frequency of HIV-specific T cells or in the ability to recognize the autologous virus, when compared to progressors. Thus far the only difference in the HIV-specific immune response that distinguishes LTNP from progressors is the maintenance of HIV-specific CD8+ T cells with a high proliferative capacity. This proliferation parallels perforin expression required for effective killing of HIV-infected CD4+ T cells. A deeper understanding of the basis of immunologic control in LTNP and the loss of immunologic control in progressors is likely to provide information that is critical for development of immunotherapies or prophylactic vaccines for HIV.[unreadable] [unreadable] In addition to the effects of the cellular immune response on restriction of HIV replication, some cells of the innate immune response are thought to play an important role in HIV disease progression. In our work on the HIV-specific cellular immune response, the largest differences in gene expression that we observe between cells of LTNP and progressors are in those altered in response to type 1 interferons. The major producers of type 1 interferons are plasmacytoid dendritic cells (PDC). Type 1 interferons such as IFN-&#945;, or other pro-inflammatory cytokines, are produced when toll-like receptors (TLR) in PDCsbind to evolutionarily conserved bacterial and viral motifs such as HIV RNA. Type 1 interferon production by PDCs is thought to play an important role in differing rates of disease progression in simian models of HIV infection. Furthermore, HIV-1 infection has been associated with changes in the number and function of PDCs including diminished frequencies in the peripheral blood and reduced production of type I interferons in response to in vitro stimulation. However, recent data suggest a paradoxical increase in production of type 1 interferons in vivo in HIV infected patients compared to uninfected controls. Using a flow-cytometric assay to detect IFN-&#61537;-producing cells within unfractionated peripheral blood mononuclear cells (PBMC), we observed that short-term interruptions of antiretroviral therapy are sufficient to result in significantly reduced IFN-&#945; production by PDCs in vitro in response to DNA (CpG A) ligands or inactivated HIV particles. The primary cause of diminished IFN-&#945; production was reduced responsiveness of PDCs to de novo stimulation, not diminished per cell IFN-&#945; production or migration of cells to lymphoid organs. Real-time PCR analysis of purified PDCs from patients prior to and during treatment interruptions revealed that active HIV-1 replication is associated with upregulation of type I IFN-stimulated gene (ISG) expression. Treatment of hepatitis C infected patients with interferon-&#945;2b and ribavirin for hepatitis C infection resulted in a profound suppression of de novo IFN-&#945; production in response to CpG A or inactivated HIV particles, similar to the response observed in HIV-infected patients. Together, these results suggest that diminished production of type I interferons in vitro by PDCs from HIV-1 infected patients may not correlate with diminished interferon production in vivo. Rather, diminished function in vitro is likely a consequence of prior activation via type I interferons or HIV virions in vivo.[unreadable] [unreadable] In addition to the cellular immune response to HIV, the humoral immune response is also examined within this project. There is a growing consensus that eliciting neutralizing antibodies (NAb) will be necessary for an effective vaccine for HIV. However, induction of a broadly cross-neutralizing antibody response has thus far not been achieved by immunization. Identification of infected individuals with broadly cross-neutralizing antibodies to HIV may provide important information regarding the titer and breadth of neutralizing antibodies and the frequency, phenotype, and specificities of HIV-specific B cells that produce these antibodies. For these reasons our laboratory has begun to screen for such patients and analyze these parameters within the sera and peripheral blood B cell compartments. We have screened more than 100 patients for sera able to neutralize diverse HIV strains. We determined the breadth and titer of these sera against a diverse array of HIV envelopes, many of which are difficult to neutralize, and identified several patient sera showing potent and broad HIV-1 neutralization. Using antibody adsorption and elution from gp120 variants, the neutralizing specificities of two broadly reactive sera were recently mapped. These patient sera were found to be highly enriched for antibodies directed at the CD4 binding site of gp120. Therefore, we conclude that these specificities are responsible for the broad neutralizing activity of these sera. These data show that it is possible for the humoral immune response to effectively target epitopes located in the gp120 CD4-binding site and mediate broad cross-neutralization. More recently, we developed a new method of labelling HIV-specific B cells, which enhances our ability to produce human monoclonal antibodies from patients with broad cross-neutralization. Further studies will be needed to determine the prevalence of these or other specificities that mediate broad cross-neutralization in sera of HIV-1infected patients.